Stable needle-shaped crystals of natamycin

ABSTRACT

The present invention describes natamycin comprising needle shaped crystals.

This application is a divisional of application Ser. No. 11/665,155filed Apr. 12, 2007 now U.S. Pat. No. 7,727,966, now allowed, which inturn is the US national phase of international applicationPCT/EP2005/055592 filed 27 Oct. 2005 which designated the U.S. andclaims benefit of EP 04105363.8, dated 28 Oct. 2004, the entire contentof which is hereby incorporated by reference.

The present invention relates to natamycin. For more than 20 years,natamycin has been used to prevent growth of mould on cheeses andsausages. Cheeses are treated by immersion in a suspension of natamycinin water or covered by an emulsion of a polymer (mostly polyvinylacetate) in water containing natamycin. Sausages are mainly treated byimmersion or by spraycoating with a suspension of natamycin in water.Usually aqueous suspensions for immersion treatments contain 0.1 to 0.2%(w/v) of natamycin, while polymer emulsions for coating purposes contain0.01 to 0.05% (w/v) of natamycin.

For the production of such suspensions or polymer emulsions, thenatamycin may be added directly to the liquids as a powder, for instanceby using the wettable powder composition known under the brand name ofDelvocid® Instant. Instead of using the powder as such, frequently aconcentrated presuspension of the natamycin in water is prepared beforeadding the natamycin to the liquids to be used for treatments. Thepurpose of preparing a presuspension is to avoid lump formation and tofacilitate homogenisation of natamycin through the treatment liquids.Mostly these methods of preparing suspensions for treating food areadequate. However, when several types of suspension with varyingconcentrations of natamycin have to be prepared or several portions oflarge quantities need to be produced over a longer period such methodsare less convenient.

When natamycin is added as a powder to treatment preparations, thelaborious weighing of the powder has to be repeated for each type ofsuspension, which multiplies the number of possible nuisances.

When a presuspension in water is used to avoid inaccurate dosages, thepresuspension has to be agitated continuously to prevent the natamycinfrom settling down or the presuspension has to be added in total to thetreatment preparations.

Aqueous suspensions of natamycin are well known in practice. Under thebrand name of Pimafucin®, sterile suspensions of natamycin arecommercially available as a 1% or a 2.5% presentation in small bottlesof up to 100 ml. Pimafucin® is mainly used for medical purposes. Torender these suspensions suitable as a multiple dose presentation, thesuspensions are preserved by using benzalkonium chloride, a preservativeof the quaternary ammonium type. Because of the sedimentation of thesolid natamycin during storage, such a suspension has to be shaken wellevery time prior to use.

As mentioned above, natamycin present in a suspension (as well as in apresuspension) tends to settle down to form a sediment at the bottom ofa container. In case the suspension is not agitated continuously asediment will be formed on the bottom which is only very difficult tobring back into the suspended state.

The objective of the invention is to provide a natamycin that does notsettle down very quickly and/or that is easy to resuspend.

DESCRIPTION

The present invention provides a new form of natamycin crystals, whichare in a needle-shaped form. According to a first aspect, the inventionrelates to natamycin comprising needle shaped crystals. Theneedle-shaped crystals of the invention are particularly well-suited tobe used in suspensions. A sediment of the crystals of the invention aremore easily brought back into the suspended state than other type ofnatamycin having other crystal type. The natamycin crystals according tothe invention have a needle or cylindrical form. Preferably, thenatamycin of the invention comprises at least 90% w/w (anhydrous basis)of needle shaped crystals as defined below. More preferably, at least95% of needle shaped crystals, even more preferably at least 95% andmost preferably at least 98%.

The mean length of needle shaped crystals will in general be between 0.1and 20 μm. Preferably, the crystals having a needle shaped form have alength of more than 2 μm, more preferably have a length of more than 4μm, most preferably have a length of more than 6 μm. Preferably, themean diameter of the needle shaped crystals is between 0.1 and 2 μm,more preferably less than 1.5 μm and most preferably a length of lessthan 1.0 μm. By “length” and “diameter” is meant the length and diameteras measured with an (Olympus) microscope (type BH-2) with a total usedmagnification of 1000 times, whereby the length is the largest lengthsize of the crystal and the diameter is the size of the thickness of thecrystal measured in the middle of the length and perpendicular to thelength direction. The mean length and the mean diameter are bothdetermined by measuring at least 100 crystals. The needle-shaped crystalform of natamycin is different from the presently known forms ofnatamycin. The known natamycin crystals have in general a plate-likeform. By plate-like is meant a form whereby the length and width are ofthe same order of dimension, which is in general 10 times higher thanthe thickness of the plate crystals.

Although the plate and needle crystals of the natamycin have acompletely different form, the crystal lattice is for both forms thesame. This crystal lattice is often indicated as Alpha-natamycin. Thepresent invention preferably relates to needle-shaped crystals ofAlpha-natamycin.

According to another aspect of the present invention, there is provideda process to produce the needle-shaped crystals of the invention. In thefirst step of this process, natamycin is dissolved in an aqueoussolution. The solution comprises at least 80 wt %, preferably at least90 wt % more preferably at least 95 wt % and still more preferably atleast 98 wt % and preferably at least 99 wt % water as solvent. Althoughat neutral pH, natamycin will hardly dissolve in water, at low pH, e.g.at a pH lower than 4.0 preferably between 1.0 and 3.0 or at high pH,e.g. at a pH above 10.0, preferably between 10.0 and 14.0, natamycinwill easily dissolve in water. Preferably, natamycin is dissolved at apH ranged between 10 and 14, more preferably at a pH ranged between 11and 13. In general, natamycin of a purity of more than 90 wt %(including hydrate) preferably of a purity of 95% is used. It is foundthat also natamycin with lower purity can be used in the process of theinvention.

In general, the natamycin solution will contain 1 to 300 g/l ofnatamycin, preferably 5 to 200 g/l and most preferably 10 to 100 g/l ofnatamycin. After that a dissolved natamycin solution has been obtained,the pH is brought to neutral. In general the pH will be brought to a pHranged between 4 and 10, preferably to a pH ranged between 5 and 9 andmore preferably between 6 and 8. Lowering the pH can for example be donewith HCl. Increasing the pH can for example with NaOH. But also othersuitable acids or bases can be used in the process of the invention.

During the pH neutralisation, the crystals according to the presentinvention are formed. During the addition of acid or base the liquid ispreferably stirred to prevent that local pH difference will occur.Typically, the addition of the acid or base to the dissolved natamycinsolution to form the suspension of natamycin at neutral pH will take 5to 50 minutes, preferably 10 to 30 minutes. We noted that shorter times,for example 1 minute does not always result in needles but more or lessin lump-like natamycin possibly due to local pH variations. Times ofmore than 1 hour are possible and suitable needles are also formed, butlonger times are economically less attractive. The temperature of thesolution or suspension is kept in general between 5° C. and 35° C.,preferably between 15° and 25° C. The crystallized needles can be driedor can be used as such. Preferably the crystals and the liquid phase arenot separated but can be used or stored as a suspension. According tothe method of the invention, preferably the solvent used in the processof the invention comprises art bast 90 wt % water. More preferably thesolvent comprises at least 95 wt % of water, even more preferably atleast 98 wt % water, even more preferably at least 99 wt % water andmost preferably 100 wt % of water. The needles formed preferablycomprise less than 5 ppm, more preferably less than 1 ppm of a solventother than water. Preferably, the crystals contain less than 5 ppm of analcohol such as methanol, more preferably less than 1 ppm of alcoholsuch as methanol.

Herein above is mentioned that preferably natamycin of more than 90 wt %purity is used. This wt % includes the hydrate or metal salt form ofnatamycin. This wt % purity is based on the compounds as obtained afterfermentation or recovery or purification of natamycin and addition ofother compounds are not included in this calculation. For example, whena 50% natamycin product is sold which also includes 50% of a filler suchas lactic acid, this filler is not included in the purity figure.

As discussed above a sediment of the needle-shaped crystals of thepresent invention can be easily brought back in the suspended statewithout exclusive stirring or shaking. Another advantage of theneedle-shaped crystals is that this crystal form is stable. For exampleit is found that after 6 months of storage of a needle-shaped crystalsuspension, which was stored at 10° C., the suspension still containedneedle-shaped crystals having the same mean length.

Another surprising effect of the crystals of the present invention isthat when the suspension is supplied to a surface to be coated with thesuspension, the surface distribution of the natamycin crystals isimproved in the sense that because of the special needle-shaped crystalscompared to plate like crystals local concentrations (on micron-scale)are more uniform.

The natamycin of the invention and any composition comprising thenatamycin of the invention can be used for any type of applications infood products, agricultural products, cosmetic products orpharmaceutical products.

According to a further aspect, the invention relates to a compositioncomprising the natamycin of the invention. The composition may be aliquid composition, a coating emulsion, a dry formulation such as apowder, a granulate or a tablet.

The amount of natamycin, in a liquid composition may be from 0.001% to2% w/w. Preferably, the amount is from 0.01% to 1% w/w. Preferably, theliquid composition is used in an immersion treatment or a sprayingtreatment. In principle, the immersion liquid may be of any kind. Whenan aqueous system is used, the addition of a surfactant may be ofadvantage, in particular for treating objects with a hydrophobicsurface.

In a coating emulsion according to the invention, the amount ofnatamycin, may be from 0.001% to 2% w/w, preferably from 0.01% to 1% w/wand more preferably from 0.01% to 0.5% w/w. The coating emulsion may beof the o/w or w/o type. Particularly preferred are emulsions preparedfrom coating emulsions commonly employed in the food industry. Forexample, for the treatment of hard cheeses an aqueous polymer emulsionof the polyvinyl acetate type may be used.

In a dry formulation according to the invention, the amount of natamycinmay be from 1% to 95% w/w, preferably from 10% to 70% w/w, morepreferably from 30% to 55% w/w.

A composition comprising natamycin can be added to the product to betreated at any moment or step of the processing. The composition can beadded in the end product before packaging, during processing or in anyingredients used to prepare the products. The advantage of the presentinvention is that it allows the production of microbiologically stableand safe products with low concentration of anti-microbials. Theinnovative composition can be used to preserve microbiological safetyand/or stability in all kind of products. Preferably the product is afood product such as dairy products, dairy desserts such as ice creammixes, yoghurt, or cottage cheese, ricotta, cream cheese, sour cream.Other preferred food products are hot-baked flour products, spreads,margarines, sauce, dressings or any other foodstuffs distributed atambient or chilled temperatures. Other preferred food products arebeverages like fruit juice, wine. Preferred food products have pHranging from pH 2.0 to pH 7.0. The addition of the composition of theinvention comprising natamycin to food products is furthermore expectedto reduce or completely eliminate moulds, yeasts and bacterial outgrowthon the food products in the time frame comprised between the end of theprocessing of the product, size reduction included and commercial sale.This time frame varies with the type of food, the distribution and saleconditions. Preferably, the product to be treated is such that itssurface will be in contact with oxygen at the end of its processingand/or later on if a reduction of size occurs.

According to another preferred embodiment, the composition comprisingthe natamycin of the invention is a composition suitable for treatingagricultural products such as fruits, vegetable and seed. Preferredfruits are citrus fruit, bananas, ananas, strawberry and the like.

According to another preferred embodiment, the composition comprisingthe natamycin of the natamycin of the invention is a pharmaceuticalpreparation suitable for topical application. Examples of suitablepharmaceutical preparations are lotions, creams and ointments.

According to another preferred embodiment, the anti-microbialcomposition of the invention comprising natamycin has a pH ranging frompH 2.0 to pH 7.0, more preferably from 3.0 to 5.0. According to anotherpreferred embodiment, the anti-microbial composition of the inventionfurther comprises water and/or salt and/or any component selected fromthe group consisting of a solvent, a surfactant, a carrier, a food acid,a thickener, any other food grade anti-microbial compound. Preferredsolvents, surfactants and carrier are already described in WO 95/08918.Preferred carrier is fumed silica. Preferred solvents are an alcoholicsolvent comprising one or more lower alcohols (C1-C4), an aqueous acid,an aqueous alkali, glacial acetic acid or mixtures of an alcoholicsolvent comprising one or more lower alcohol with either an aqueousacid, an aqueous alkali or glacial acetic acid. Preferred surfactantsare sodium lauryl sulphate, dioctyl sulphosuccinate, calcium chloride,or surfactants of the non-ionic types, for example those which are knownunder the brand names Tween, Span, Brij, and Myrj. Preferred thickenersmay be any thickener known in the art for use in food products.Preferred thickeners are hydroxypropylmethylcellulose (HPMC), and/orgums, and/or carrageenan and/or methylcellulose. Preferred gums arexanthan gum and/or gellan gum and/or arabic gum as described in U.S.Pat. No. 5,962,510 or U.S. Pat. No. 5,552,151. Preferred food acids areorganic acidic anti-fungal agent such as benzoic acid, propionic acid orsorbic acid or other acids such as acetic acid or lactic acid asdescribed in EP 608 944 B1.

According to a further aspect of the invention, a product is providedwhich has been treated with the natamycin of the invention or with thecomposition of the invention including the natamycin of the invention.

According to another preferred embodiment, the product is awater-containing product for example a food product, which comprises thenatamycin of the invention or the composition of the invention includingnatamycin.

According to a further aspect of the invention, there is provided theuse of the natamycin of the invention to obtain a product, for example afood product, comprising the composition of the invention includingnatamycin.

According to a further aspect of the invention, there is provided amethod for treating a product, for example a food product, with thenatamycin of the invention or which comprises the composition of theinvention including the natamycin of the invention.

According to a further aspect, the invention relates to a method forprotecting a product against fungal spoilage by applying the natamycinof the invention or the composition of the invention including thenatamycin of the invention.

Before, during or after the addition of natamycin to the product, otheringredients such as colorants, texturals etc. can be added as well tothe product.

The invention will further now be illustrated by examples, which shouldnot be construed as limiting the scope of the invention.

EXAMPLES Material and Methods

Materials

Delvocid® containing 50% active (w/w) natamycin, DSM Food Specialties,(P.O. Box 1, 2600 MA, Delft, The Netherlands).

Hydrochloric acid, purchased from Gaches Chimie France, (31750Escalquens, France).

Sodium hydroxide, purchased from Gaches Chimie France, (31750Escalquens, France).

Example 1

This example describes a method to prepare a concentrate dispersion ofnatamycin with needle shaped crystals suitable to be used as testmaterial in direct comparison to standard natamycin crystals.

A suspension of natamycin was prepared by suspending 150 gram Delvocid®in 4000 gram water while stirring with an electric top stirrer, type RW20 DZM, from Janke & Kunkel equipped with a rushton stirrer. Whilestirring, 600 ml 4M NaOH solution is added to a final pH of ±12resulting in a clear light yellow liquid. Directly after obtaining aclear liquid, 600 ml 4M HCl solution was added in 20 minutes time to afinal pH of 6.5. During addition of the HCl, a haze appeared and finallythe liquid changed to a very viscous suspension.

This suspension was used as starting material for the other describedexamples. We have found that the use of distilled or tap water (Delft,Holland or St. Clar (near Toulouse) France) did not have an influence onthe needle formation.

Example 2

This example describes another method to prepare a concentratedispersion of natamycin with needle shaped crystals. A suspension ofnatamycin was prepared by suspending 150 gram Delvocid® in 4000 gramwater while stirring with an electric top stirrer, type RW 20 DZM, fromJanke & Kunkel equipped with a rushton stirrer. While stirring, 4M HCLwas added to a final pH of 0.8 resulting in a clear light yellow liquid.Directly after obtaining a clear liquid, 4M NaOH was added slowly to afinal pH of ±6.5. During addition of the NaOH, a haze appeared andfinally the liquid changed to a very viscous suspension. The obtainedcrystals had similar characteristics as the crystals obtained in Example1.

Example 3

This example describes the method that was used to analyze the amount ofactive natamycin in a water-based mixture of several components.

The method used was a HPLC analysis based on the International DairyFederation (Provisional IDF Standard 140, 1992) with a Lichrosorb RP 8column.

Detection was at 303 nm UV with a range of 0.1-4 mg/L with an injectionvolume of 20 μl. Sample preparation was carried out by weighing 2 g ofprepared formulation with an accuracy of 1 mg in a measuring flask. 4 mldemineralized water (demiwater) was added and the mixture was stirredfor 15 minutes to get a homogeneous suspension. Subsequently, 80 mlethanol was added and the mixture was stirred for 10 minutes. Afterultrasonic treatment, the solution was filled up to 100 ml withdemiwater and then diluted and/or filtered (0.2 μm) before injecting.The amount of active natamycin was calculated as ppm against a series ofstandards.

Example 4

This example describes the test performed to analyze the sedimentationdifferences.

1.5 g Delvocid® was added to 998.5 gram tapwater and dispersed well byan electric top stirrer, type RW 20 DZM, from Janke & Kunkel equippedwith a rushton stirrer.

1.5 gram of Delvocid® made according to Example 1 was mixed withtapwater to a final weight of 1000 gram by an electric top stirrer, typeRW 20 DZM, from Janke & Kunkel equipped with a rushton stirrer.

Both homogeneous mixtures were poured in glass tubes and observed intime on demixing.

The results are set out in Table 1.

TABLE 1 Time (minutes) Standard natamycin Recrystallized natamycin 0Homogeneous Homogeneous 40 Start demixing observed Homogeneous 60 Twolayers observed Homogeneous 110 Pellet formation Homogeneous 180 Pelletformation Homogeneous 4320 Pellet and clear top layer Start demixingobserved

Example 5

This example describes the test that was performed to analyze theresuspendability of settled natamycin in a low viscous dispersion. 1.5gram Delvocid® was added to 998.5 gram tapwater and dispersed well by anelectric top stirrer, type RW 20 DZM, from Janke & Kunkel equipped witha rushton stirrer. An amount equal to 1.5 gram Delvocid® made accordingto Example 1 was mixed with tapwater to a final weight of 1000 gram byan electric top stirrer, type RW 20 DZM, from Janke & Kunkel equippedwith a rushton stirrer. Both homogeneous mixtures were poured in glasstubes and not touched for 26 days. After 26 days, the glass tubes wereturned around once and it was observed that the pellet of therecrystallized natamycin was almost completely resuspended and that thepellet of the standard natamycin remained a solid pellet.

Example 6

This example describes the stability of recrystallized natamycin, anatamycin dispersion is made according to Example 1 and followed in timeon stability.

The rest activity of natamycin is set out in Table 2.

TABLE 2 Incubation time (months) Found natamycin activity (%) 0 100 1100 2 99 4 100 6 98

The stability of the recrystallised natamycin is very high for at leastsix months.

The invention claimed is:
 1. Crystals of α-natamycin of which at least90% w/w are needle shaped crystals having a mean length of between 0.1and 20 μm.
 2. The α-natamycin crystals according to claim 1, wherein atleast 95% w/w of the crystals are needle shaped crystals.
 3. Theα-natamycin crystals according to claim 1, wherein at least 98% w/w ofthe crystals are needle shaped crystals.
 4. The α-natamycin crystalsaccording to claim 1, wherein 60% of the needle shaped crystals have alength of more than 2 μm.
 5. The α-natamycin crystals according to claim1, wherein at least 90% w/w are needle shaped crystals having a meandiameter of between 0.1 and 2 μm.
 6. The α-natamycin crystals accordingto claim 1, wherein 60% of the needle shaped crystals have a diameter ofless than 2 μm.
 7. A liquid suspension composition comprising theα-natamycin crystals according to claim
 1. 8. The liquid suspensioncomposition according to claim 7, wherein the amount of α-natamycinpresent in said composition is from 0.001% to 2% w/w.
 9. The liquidsuspension composition according to claim 7, wherein the amount ofα-natamycin present in said composition is from 0.01% to 1% w/w.
 10. Theliquid suspension composition according to claim 7, wherein thecomposition has a pH ranging from 2.0 to 7.0.
 11. The liquid suspensioncomposition according to claim 7, wherein the composition has a pHranging from 3.0 to 5.0.
 12. The liquid suspension composition accordingto claim 7, wherein the composition further comprises a componentselected from the group consisting of a solvent, a salt, a surfactant, acarrier, a food acid, a thickener and any other food grade antimicrobialcompound.
 13. A composition comprising the α-natamycin crystalsaccording to claim 1, wherein said composition is not a liquidcomposition.
 14. A coating emulsion composition comprising theα-natamycin crystals according to claim
 1. 15. The coating emulsioncomposition according to claim 14, wherein the amount of α-natamycinpresent in said composition is from 0.001% to 2% w/w.
 16. The coatingemulsion composition according to claim 14, wherein the amount ofα-natamycin present in said composition is from 0.01% to 1% w/w.
 17. Thecoating emulsion composition according to claim 14, wherein the amountof α-natamycin present in said composition is from 0.01% to 0.5% w/w.18. The coating emulsion composition according to claim 14, wherein thecomposition has a pH ranging from 3.0 to 5.0.
 19. The coating emulsioncomposition according to claim 14, wherein the emulsion is of theoil-in-water type.
 20. The coating emulsion composition according toclaim 14, wherein the emulsion is of the water-in-oil type.
 21. A dryformulation composition comprising the α-natamycin crystals according toclaim
 1. 22. The dry formulation composition according to claim 21,wherein the amount of α-natamycin present in said composition is from 1%to 95% w/w.
 23. The dry formulation composition according to claim 21,wherein the amount of α-natamycin present in said composition is from10% to 70% w/w.
 24. The dry formulation composition according to claim21, wherein the amount of α-natamycin present in said composition isfrom 30% to 55% w/w.
 25. The dry formulation composition according toclaim 21, wherein said dry formulation composition is selected from thegroup consisting of a powder, a granule, and a tablet.
 26. A producttreated with the α-natamycin crystals according to claim
 1. 27. Theproduct according to claim 26, wherein the product is selected from thegroup consisting of a food product, an agricultural product, a cosmeticproduct and a pharmaceutical product.
 28. A product treated with theliquid suspension composition according to claim
 7. 29. A method fortreating a product with the α-natamycin crystals of claim 1, comprisingapplying the α-natamycin crystals of claim 1 to said product.
 30. Themethod for treating a product according to claim 29, wherein saidproduct treated is a food product or an agricultural product.
 31. Themethod for treating a product according to claim 30, wherein the producttreated is a food product and said food product is selected from thegroup consisting of dairy products, hot-baked flour products, spreads,margarines, sauces, dressings, fruit juices, and wines.
 32. The methodfor treating a product according to claim 30, wherein the producttreated is an agricultural product and said agricultural product isselected from the group consisting of fruits, vegetables, and seed. 33.A method for protecting a product against fungal spoilage, comprisingapplying the α-natamycin crystals of claim 1 to said product.